Not applicable.
The present invention relates generally to methods for preventing the growth of specific cells in a multi-cellular eukaryote, particularly plant cells. Recombinantly modified plant cells for expression of cytotoxic genes are also provided.
One key objective of plant genetic engineering is to create novel traits either through expression of an introduced gene or by silencing of an endogenous gene. One use of targeted gene expression is the elimination of specific plant cells through the production of an enzyme that is lethal to the cell. In order to eliminate only a specific set of cells, it is necessary that expression of a potentially lethal function be controlled precisely such that the cell-lethal function is expressed only in the cells targeted for elimination and in no others.
Several different approaches have now been attempted to create novel plant traits using a single component cell lethality system. In one-component cell lethality systems, specific cell types are targeted for elimination using a single promoter driving expression of a cytotoxic gene product. These approaches are initiated by the characterization of promoters that are active in specific tissues or under specific conditions. For example, male sterility has been demonstrated using promoters active in tapetal tissue. A number of different promoters have been identified that are expressed in tapetal tissue and other tissue. A further example is the use of cell lethality to create disease resistance via a hypersensitive response. A number of promoters have been characterized by different workers that are induced upon pathogen infection. A further example is the attempt to create nematode resistance by killing giant cells, the specific root cells upon which cyst and root knot nematodes feed. A number of promoters have been characterized by different workers that are induced in giant cells, but again sufficient promoter specificity has been difficult to achieve.
In one example (Strittmatter et al., Bio/Technology 13:1085-90 (1995)), the workers cite the difficulty of creating transgenic plants using a construct containing a ribonuclease coding sequence (obtained from Bacillus amyloliquifaciens, barnase) under the control of a promoter induced upon infection with the fungus Phytophthora infestans. Their solution to the difficulty was to express a protective function, barstar, under the control of a constitutive promoter that would hopefully protect non-infected cells, but allow infected cells to be killed. A further example is the attempt to create nematode resistance by killing the specific root cells upon which cyst and root knot nematodes feed, as described in WO 92/21757, WO 93/10251, WO 93/18170, WO 94/10320, and WO 94/17194. A number of promoters have been characterized by different workers that are induced in specialized nematode feeding cells, but again sufficient promoter specificity has been difficult to achieve.
In one case (WO 93/10251), the difficulty of obtaining sufficient promoter specificity is addressed through expression of a protective function in cells other than the target cells. Another example of the protective approach is described in WO 96/26283, which described the production of male sterility using the tapetal specific promoter TA29 from tobacco to program expression of barnase. As in the example above, the protective function for the barnase protein is the barstar protein, whose expression is sought in non-target tissues. Unfortunately, for many potentially useful cell-lethal functions, protective functions are not available. In order to make a protective approach work, it is necessary to identify a second promoter with the requisite xe2x80x9cinversexe2x80x9d specificity.
In vertebrates similar cell lethality approaches have been reported to ablate specific cell types or tissue types as an experimental tool, or to kill cells involved in a disease state, such as HIV-infected cells or metastatic cancer cells. The principal cell lethality function chosen for cell ablation is the diphtheria toxin (DT) A chain, which adenoribosylates elongation factor EF-2, thus blocking protein synthesis. Herrera et al., Proc. Natl. Acad. Sci., USA 91:12999-13003 (1994). Because of the extreme toxicity of the DT A chain, precise expression is critical. An approach that has been taken in therapeutic situations is the specific introduction and/or expression of a thymidine kinase (tk) gene. The tk gene product is a conditional cell-lethal function, requiring the presence of a nucleoside analog such as ganciclovir for lethality. Brady et al., Proc. Natl. Acad. Sci., USA 91:365-69 (1994) describe the use of this approach for specific ablation of human immunodeficiency virus Tat-expressing cells following introduction of a tk gene whose expression is under control of the Tat protein and treatment with ganciclovir.
In developing ways to kill specific subpopulations of cells within an organism, such as metastatic cancer cells in a mammal, the requirement of xe2x80x9ctwofold specificityxe2x80x9d, has been recognized. Panchal et al., Nature Biotechnol. 14:852-56 (1996). The approach taken by Panchal et al. was to use immunorecognition of the surface of cancer cells as the first level of specificity, and specific protease activities of cancer cells as the second level of specificity.
What is needed in the art are compositions and methods which provide selective elimination or inhibition of growth of a selected cell type in an organism, for example, in a plant. The present invention provides these and other advantages.
In one aspect, the present invention is directed to a plant cell. The plant cell comprises a first expression cassette comprising a first non-constitutive plant promoter operably linked to a polynucleotide encoding a first polypeptide and a second expression cassette comprising a second non-constitutive plant promoter operably linked to a polynucleotide encoding a second polypeptide. At least the first or the second expression cassette is heterologous to the cell. Further, the first and second promoters have different but overlapping specificities such that the first and second polypeptides are expressed in the same cell.
In some embodiments, the presence of the first and second polypeptides in the same cell impairs cellular function. In some embodiments, the first and second polypeptides each comprise a separate subsequence of a single functional polypeptide. The functional polypeptide can be a ribonuclease such as Barnase, or T1. The functional polypeptide be modified to have enhanced stability. In one embodiment, the enhanced stability barnase is bn3-2 and bn5-2. In additional embodiments of the plant cell, the first polypeptide is an avirulence gene product derived from a plant pathogen and the second polypeptide is a resistance gene product associated with the avirulence gene.
For example, first polypeptide can be avr9 and the second polypeptide Cf9. The functional polypeptide can be a nuclease or colicin. In some plant cell embodiments, the first or the second promoter is a tissue-specific promoter such as when each is functional in seeds or tapetal cells. In some embodiments, the first or second promoter is induced following interaction with a plant pathogen or pest.
In another aspect, the present invention relates to a plant cell comprising a first expression cassette comprising a first plant promoter operably linked to a polynucleotide encoding a first polypeptide and a second expression cassette comprising a second plant promoter operably linked to a polynucleotide encoding a second polypeptide. The first and second polypeptides each comprise a separate subsequence of a single functional polypeptide.
Often, the functional polypeptide impairs cellular function. In some embodiments, the first and second promoters have different but overlapping specificities such that the first and second polypeptides are expressed in the same cell.
In another aspect, the present invention relates to a method for modifying the cellular function of a plant cell. The method comprises the steps of introducing into the cell a first expression cassette comprising a first non-constitutive plant promoter operably linked to a polynucleotide encoding a first polypeptide and a second expression cassette comprising a second non-constitutive plant promoter operably linked to a polynucleotide encoding a second polypeptide, wherein the first and second promoters have different but overlapping specificities such that the first and second polypeptides are expressed in the same cell. The first or the second expression cassettes can be introduced into the plant cell through a sexual cross.
In yet another aspect, the present invention relates to a method of modifying cellular function in a plant cell. The method comprises the steps of introducing into a plant cell a first expression cassette comprising a first plant promoter operably linked to a polynucleotide encoding a first non-functional polypeptide and a second expression cassette comprising a second plant promoter operably linked to a polynucleotide encoding a second non-functional polypeptide, wherein the first and second polypeptides each comprise a separate subsequence of a single functional polypeptide. Often, the first and second promoters have different but overlapping specificities such that the first and second polypeptides are expressed in the same cell.
In a further aspect, the present invention is directed to a method for preventing the growth of a eukaryotic cell. The method comprises the steps of introducing into the cell, a first expression cassette comprising a first tissue-specific plant promoter operably linked to a polynucleotide encoding a first polypeptide and a second expression cassette comprising a second tissue-specific plant promoter operably linked to a polynucleotide encoding a second polypeptide, wherein the first and second promoters are functional in the cell and presence of the first and second polypeptides in a cell impairs cellular function. In some embodiments the cell is a mammalian cell. The mammalian cell can be in a non-human animal. Often, the first and second expression cassettes are introduced into the cell using a retroviral vector.